CN109030663A - A kind of method that UHPLC method detects chemical component in Brown Mixtura - Google Patents
A kind of method that UHPLC method detects chemical component in Brown Mixtura Download PDFInfo
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Abstract
The invention discloses the method for chemical component in a kind of UHPLC method detection Brown Mixtura, the method for chemical component includes detection Brown Mixtura chemical component and detection Brown Mixtura blood plasma Metabolite in the UHPLC method detection Brown Mixtura.Detection method runing time is short, and has good separating degree to numerous components in Brown Mixtura sample.Further, this method has good precision, repeatability and stability, and the chemical component suitable for Brown Mixtura detects, and is suitable for the detection of Brown Mixtura blood plasma Metabolite simultaneously.Ingredient in Brown Mixtura sample can be carried out rapidly, comprehensively detecting, there is good separating degree to numerous components in Brown Mixtura sample；Especially be able to detect that in the second test solution it is complicated it is micro enter blood component, and separating degree is good.Therefore, it is detected using method of the invention, is conducive to illustrate the component in Brown Mixtura sample comprehensively.
Description
Technical field
The invention belongs to pharmaceutical technology fields, further belong to medical detection technique field, and in particular to a kind of UHPLC method
The method for detecting chemical component in Brown Mixtura.
Background technique
The compound that Brown Mixtura is made of Radix Glycyrrhizae extractum powder, opium powder, camphor, oil of badian, sodium benzoate etc.
Preparation is recorded in Chinese Pharmacopoeia 2015 version two, is protectiveness antitussive, clinically for general cough and on exhale
The treatment of cough for inhaling road infection, acute tracheitis initial stage etc., can be effectively relieved symptom.
Modern research shows that the main component in Brown Mixtura is alkaloids, flavonoids and ter penoids, it can be pre-
The effect of sore-throat and cough after anti-Endotracheal Intubation Under General Anesthesia, it is also possible to which the maturity state by adjusting dendritic cells alleviates
The reaction of air flue, to have the function that kobadrin.
The detection method for establishing suitable chemical component is that the importance of quality control is carried out to drug.It discloses at present
The detection method of the chemical component of a variety of Brown Mixturas, but there is not yet inspection for metabolin in the blood plasma of Brown Mixtura
Survey method.
The detection method of chemical component about Brown Mixtura, Sun Guoxiang etc. are disclosed with HPLC finger-print to compound
Licorice tablet implement total quality control method (HPLC finger-print to Brown Mixtura implement total quality control method, Central-South pharmacy,
03 phase in 2008), using double qualitative double quantitative similarities as evaluation index, establish Brown Mixtura HPLC finger-print
Total quality control method.This method uses RP-HPLC method with Century SIL C18BDS column (200mm × 4.6mm, 5 μm);With
Fingerprint spectrum Fr is that index optimization selects finger-print testing conditions, determines that mobile phase is 1% acetic acid water -1%
The elution of acetate acetonitrile low pressure gradient, ultraviolet detection wavelength: 254nm, column temperature: (30.00 ± 0.15) DEG C, sample volume: 5 μ L.The party
The established HPLC finger-print of method has preferable precision and reproducibility, and the quality suitable for Brown Mixtura controls, and recognizes
It is most reasonable, the most objective finger-print of macro qualitative analysis quantitative assessment Chinese materia medica preparation quality for dual qualitative and dual quantitative similarity method
Assessment technique.
Yang Hongjuan etc. discloses RP-HPLC while measuring the method for 4 component contents in Brown Mixtura (Yang Hongjuan etc., RP-
HPLC measures the content of 4 ingredients in Brown Mixtura, Pharmaceutical Analysis magazine, 01 phase in 2013 simultaneously), use efficient liquid phase
The content of chromatography for simultaneous detection Morphine in Compound Liguoric Tablets, codeine phosphate, liquiritin and glycyrrhizic acid is specific to use
PhenomenexLuna-C18 (250 mm × 4.6 mm, 5 μm) chromatographic column, it is molten with -0.02 molL-1 potassium dihydrogen phosphate of acetonitrile
Liquid (phosphoric acid tune pH to 4.0) is mobile phase, and gradient elution, 1.0 mLmin-1 of flow velocity, Detection wavelength is 220 nm (0~23
Min detects morphine and codeine), 254 nm (23~50 min detect liquiritin and glycyrrhizic acid), column temperature is 35 DEG C.
Zhao Yue so etc. disclose HPLC method while measuring liquiritin in Brown Mixtura, isoliquiritin, glycyrrhizin, glycyrrhizic acid
Method (HPLC method measures liquiritin in Brown Mixtura, isoliquiritin, glycyrrhizin, glycyrrhizic acid simultaneously, Chinese patent drug, 2014
04 phase).This method is using Dikma Diamonsil C18 (250 mm × 4.6 mm, 5 μm) chromatographic column;Mobile phase is acetonitrile-
0.05% phosphate aqueous solution, gradient elution;Detection wavelength is 276 nm (0 ~ 16 min), 360 nm (16 ~ 24 min), 276 nm
(24~28 min),254 nm(28~38 min);1.0 mL/min of volume flow;30 DEG C of column temperature.
Establish quality control of the detection method for drug, the pharmacology, drug effect, in vivo metabolism and medicine of suitable chemical component
Effect material base etc. research and application are of great significance.The chemical component detection method of existing Brown Mixtura is mostly
High performance liquid chromatography, retention time is longer, at least needs 40 minutes or even a percentage clock.In addition, existing method is limited to more
The assay of one or several index components in Brown Mixtura, it is difficult to which the chemical component in Brown Mixtura is carried out
It illustrates comprehensively.In addition, the prior art cannot the blood plasma Metabolite to Brown Mixtura detect.Therefore a kind of energy solution is developed
Certainly the detection method of above-mentioned technical problem is very important.
Summary of the invention
The purpose of the present invention is to provide the methods of chemical component in a kind of UHPLC method detection Brown Mixtura sample.
The object of the present invention is achieved like this, the method for chemical component in the UHPLC method detection Brown Mixtura
Including detection Brown Mixtura chemical component and detection Brown Mixtura blood plasma Metabolite, specifically includes the following steps:
A, prepared by test solution:
Brown Mixtura is taken to carry out that the first test solution is prepared；The blood plasma of Brown Mixtura Metabolite is taken to be prepared
Obtain the second test solution；
Preparation method specifically:
1) the first test solution: taking Brown Mixtura, crushes, and 10 ~ 40 times of glycyrrhiza compound sheet weight of organic solvent is added and mentions
It takes, extracting solution filtering, filtrate is the first test solution；
2) the second test solution: by the solid-phase extraction column after being activated on the blood plasma containing Brown Mixtura Metabolite, successively
It being eluted with water and methanol, collects meoh eluate, volatilize, the acetonitrile solution that percentage by volume 10% is added redissolves, vortex 3 ~
10 ~ 30min is centrifuged after 5min, taking supernatant is the second test solution；
B, prepared by reference substance solution: reference substance solution is glycyrrhizin, isoliquiritigenin, enoxolone, liquiritin, isoliquiritin, sweet
Careless chalcone B, celery sugar liquiritin, licochalcone A, glabridin, ononin, secondary liquorice chalcone, onocerin and camphor tree
One or more of brain reference substance；
Preparation method be each reference substance is accurately weighed, solubilizer be made every kind of reference substance concentration be 10 ~ 500 μ g mL-1 pair
According to product solution；The solvent is 50 ~ 100% methanol aqueous solution of percentage by volume, 50 ~ 100% ethanol waters or 50 ~ 100% second
Nitrile aqueous solution；
C, UHPLC is measured:
UHPLC chromatographic condition is as follows:
Chromatographic column: C18Column；Mobile phase: 0.1% aqueous formic acid (A)-acetonitrile (B)；Gradient elution: 0 min, 5% B；10
Min, 42% B；15 min, 46% B；20 min, 55% B；25 min, 65% B；30 min, 100% B.Flow velocity is 0.3 mL
min-1；Sample volume 0.5-20 μ L.
Detection method runing time is short, and has good separation to numerous components in Brown Mixtura sample
Degree.Further, this method has good precision, repeatability and stability, and the chemical component suitable for Brown Mixtura is examined
It surveys, and is suitable for the detection of Brown Mixtura blood plasma Metabolite simultaneously.Ingredient in Brown Mixtura sample can be carried out
Rapidly, it comprehensively detects, there is good separating degree to numerous components in Brown Mixtura sample；Especially it is able to detect that
In second test solution it is complicated it is micro enter blood component, and separating degree is good.Therefore, it is examined using method of the invention
It surveys, is conducive to illustrate the component in Brown Mixtura sample comprehensively.
Numerous components in Brown Mixtura sample can be effectively separated using UHPLC method of the present invention, and into one
The component in Brown Mixtura sample that step can separate UHPLC method using above-mentioned Mass Spectrometry Conditions is effectively detected, and is detected
High sensitivity, and the ingredient Multi-level information obtained is abundant, can be realized and is accurately analyzed and identified to ingredient.Using the present invention
UHPLC-MS detection method, can identify 50 or more compounds in Brown Mixtura sample.In an embodiment
In, the first test solution is detected, identifies 55 compounds, including 42 flavones ingredients, 9 ter penoids altogether
And 4 composition of alkaloids.In another embodiment, the second test solution is detected, identifies 26 altogether
Compound, including 20 prototype ingredients and 6 metabolites.
Detailed description of the invention
Fig. 1 is that UHPLC-LTQ-Orbitrap analyzes Brown Mixtura total ion current figure under positive ion mode:
Wherein, A is 13 reference substance solutions, and B is Brown Mixtura, and C is plasma sample.
Specific embodiment
Below with reference to embodiment and attached drawing, the present invention is further illustrated, but is not subject in any way to the present invention
Limitation, based on present invention teach that it is made it is any transform or replace, all belong to the scope of protection of the present invention.
The method of chemical component includes detection Brown Mixtura chemical component in the UHPLC method detection Brown Mixtura
With detection Brown Mixtura blood plasma Metabolite, specifically includes the following steps:
A, prepared by test solution:
Brown Mixtura is taken to carry out that the first test solution is prepared；The blood plasma of Brown Mixtura Metabolite is taken to be prepared
Obtain the second test solution；
Preparation method specifically:
1) the first test solution: taking Brown Mixtura, crushes, and 10 ~ 40 times of glycyrrhiza compound sheet weight of organic solvent is added and mentions
It takes, extracting solution filtering, filtrate is the first test solution；
2) the second test solution: by the solid-phase extraction column after being activated on the blood plasma containing Brown Mixtura Metabolite, successively
It being eluted with water and methanol, collects meoh eluate, volatilize, the acetonitrile solution that percentage by volume 10% is added redissolves, vortex 3 ~
10 ~ 30min is centrifuged after 5min, taking supernatant is the second test solution；
B, prepared by reference substance solution: reference substance solution is glycyrrhizin, isoliquiritigenin, enoxolone, liquiritin, isoliquiritin, sweet
Careless chalcone B, celery sugar liquiritin, licochalcone A, glabridin, ononin, secondary liquorice chalcone, onocerin and camphor tree
One or more of brain reference substance；
Preparation method be each reference substance is accurately weighed, solubilizer be made every kind of reference substance concentration be 10 ~ 500 μ g mL-1 pair
According to product solution；The solvent is 50 ~ 100% methanol aqueous solution of percentage by volume, 50 ~ 100% ethanol waters or 50 ~ 100% second
Nitrile aqueous solution；
C, UHPLC is measured:
UHPLC chromatographic condition is as follows:
Chromatographic column: C18Column；Mobile phase: 0.1% aqueous formic acid (A)-acetonitrile (B)；Gradient elution: 0 min, 5% B；10
Min, 42% B；15 min, 46% B；20 min, 55% B；25 min, 65% B；30 min, 100% B.Flow velocity is 0.3 mL
min-1；Sample volume 0.5-20 μ L.
Step A 1) described in organic solvent be that 50 ~ 100% methanol aqueous solution of percentage by volume, 50 ~ 100% ethyl alcohol are water-soluble
Liquid or 50 ~ 100% acetonitrile solutions.
Step A 1) described in organic solvent be 60 ~ 80% methanol aqueous solution of percentage by volume, 60 ~ 80% ethanol waters
Or 60 ~ 80% acetonitrile solution.
Step A 1) described in organic solvent be 70% methanol solution of percentage by volume.
Step A 2) described in the blood plasma containing Brown Mixtura Metabolite be to be prepared by the following, specially
Animal or population clothes give Brown Mixtura, and blood sampling is placed in the anticoagulant EP pipe of heparin sodium after drug enters blood, are uniformly mixed, from
The heart takes supernatant to obtain.
Step A 2) described in activation solid-phase extraction column be successively with first alcohol and water activate.
Step A 2) described in centrifugal condition be 4 DEG C of temperature, revolving speed 14000rpm.
Organic solvent described in step B be 80 ~ 100% methanol aqueous solution of percentage by volume, 80 ~ 100% ethanol waters or
80 ~ 100% acetonitrile solutions.
The method of chemical component, further includes mass spectroscopy detection step in the UHPLC method detection Brown Mixtura, i.e., described
UHPLC method detection Brown Mixtura in chemical component method be UHPLC-MS method detect.
Mass Spectrometry Conditions are as follows: positive ion detection mode: capillary temperature: 350 °C；Sheath gas: 40.0 arb；Assist gas
Flow velocity: 20.0 arb；Spray voltage: 4 kV；Capillary voltage: 25 V；Pipe lens voltage: 110 V；Sample carries out FS using FT
Scanning, resolution ratio 30000, scanning rangem/z100~1000, width 2 is isolated；Second level and three-level mass spectrum use data dependence
Property scanning (data dependent scan, DDS), choose highest 3 peaks of upper level abundance and carry out collision induced dissociation
The scanning of (collision induced dissociation, CID) fragment, normalization collision energy are 35%, are beaten and are taken with ion trap
Detect fragment ion in pole.
The method of chemical component, concrete operations are as follows in UHPLC method detection Brown Mixtura of the present invention:
UHPLC chromatographic condition is as follows:
Chromatographic column: C18Column；Mobile phase: 0.1% aqueous formic acid (A)-acetonitrile (B)；Gradient elution: 0 min, 5% B；10
Min, 42% B；15 min, 46% B；20 min, 55% B；25 min, 65% B；30 min, 100% B.Flow velocity is 0.3 mL
min-1；Sample volume 0.5-20 μ L.In the present invention, chromatographic column can select common C18 column, preferably ACQUITY in field
UHPLC BEH C18(1.7 μm, 2.1 × 100mm).Sample volume is preferably 1-5 μ L, more preferably 2 μ L.
It should be noted that " Brown Mixtura sample " described herein is the first test solution or the second test sample
Solution, the first test solution refer to the sample obtained after Brown Mixtura solvent extraction；Second test solution refers to multiple
Plasma sample after square licorice tablet oral absorption.
The method of the present invention runing time is short, and has good separating degree to numerous components in Brown Mixtura sample；
Especially be able to detect that in the second test solution it is complicated it is micro enter blood component, and separating degree is good.Therefore, using this
The method of invention is detected, and is conducive to illustrate the component in Brown Mixtura sample comprehensively.
In the present invention, the preparation method of the first test solution: taking Brown Mixtura, crushes, and glycyrrhiza compound slice weight is added
The 10-40 times of solvent extraction measured is measured, extracting solution filtering, filtrate is test solution.It is water-soluble that the solvent is selected from 50-100% methanol
Liquid, 50-100% ethanol water, 50-100% acetonitrile solution, preferably 60-80% methanol aqueous solution, 60-80% ethyl alcohol are water-soluble
Liquid, 60-80% acetonitrile solution, more preferably 70% methanol aqueous solution.The dosage of the solvent is the 10- of glycyrrhiza compound sheet weight
40 times of amounts, preferably 15-30 times is measured, and more preferably 18-25 times is measured, most preferably 20 times amounts.The extracting method is preferably super
Sound method；Extraction time is preferably 5-40min, more preferably 10-30min, is further preferably 15-20min.It is preferred real according to one
Mode is applied, Brown Mixtura sample is made with the following method: taking Brown Mixtura, ground, 20 times of amount 70% methanol ultrasounds are added
20 min, filtering take subsequent filtrate, cross 0.22 μm of miillpore filter and obtain.
In the present invention, the preparation method of the second test solution: will be living on the blood plasma containing Brown Mixtura Metabolite
Solid-phase extraction column after change is successively eluted with water and methanol, is collected meoh eluate, is volatilized, and it is multiple that 10% acetonitrile solution is added
It is molten, it is vortexed, centrifugation takes supernatant as the second test solution.According to a preferred embodiment, successively with 5 volumes
The water activated solid extraction column of methanol and 5 volumes, is then added the blood plasma of 1 volume, finally with the water of 5 volumes and 3 volumes
Methanol elution, collect meoh eluate, at room temperature with being dried with nitrogen, the 10% acetonitrile solution redissolution of 0.1 volume is added in residue,
It is vortexed after 4 min and is centrifuged 20 min(14000 rpm, 4 DEG C), take supernatant as the second test solution.
Wherein, the blood plasma containing Brown Mixtura Metabolite can obtain by the following method: animal is oral to give compound
Licorice tablet, blood sampling is placed in the anticoagulant EP pipe of heparin sodium after drug enters blood, is uniformly mixed, and centrifugation takes supernatant, obtains the second confession
Test sample solution.In the application, the animal can for routine experiment animal, such as mouse, rat, cavy, rabbit, dog, monkey etc.,
It can be the mankind, be not particularly limited.Second test solution can be placed in -20 DEG C of refrigerators and save backup.
Compared with drug itself, chemical composition change is big after drug enters blood, and interference is more, and the detection method of drug itself is to it
The detection method referentiability of Metabolite is poor.Present inventor discovery, when to Brown Mixtura blood plasma use the above method
After being handled, when the second obtained test solution is detected using UPLC method of the invention, each component therein can
Realize good separation.
Above-mentioned UHPLC detection method can also include the steps that reference substance solution sample introduction, and wherein reference substance solution can be with
Contain glycyrrhizin, isoliquiritigenin, enoxolone, liquiritin, isoliquiritin, liquorice chalcone B, celery sugar liquiritin, Radix Glycyrrhizae Cha Er
Ketone A, glabridin, ononin, secondary liquorice chalcone, at least one of onocerin and camphor reference substance, preferably 2 kinds with
On, more preferable 5 kinds or more, further preferably 8 kinds or more.The preparation method of the reference substance solution can be with are as follows: each reference substance is accurate
Weighed, it is 10-500 μ gmL that every kind of reference substance concentration, which is made, in solubilizer-1Reference substance solution.Solvent can be selected from 50-
100% methanol aqueous solution, 50-100% ethanol water, 50-100% acetonitrile solution, preferably 80-100% methanol aqueous solution,
80-100% ethanol water, 80-100% acetonitrile solution, more preferably methanol or acetonitrile.The concentration of every kind of reference substance solution can
Think 50-200 μ gmL-1, more preferably 80-150 μ gmL-1, more preferably 100 μ gmL-1.Using above-mentioned control
Product solution sample introduction, is conducive to the identification of each component in Brown Mixtura sample.
The present invention also provides a kind of UHPLC-MS detection method of chemical component for Brown Mixtura sample,
UHPLC method is as described above, Mass Spectrometry Conditions are as follows:
Positive ion detection mode: capillary temperature: 350 °C；Sheath gas: 40.0 arb；Secondary air speed: 20.0 arb；Spray
Mist voltage: 4 kV；Capillary voltage: 25 V；Pipe lens voltage: 110 V；Sample carries out FS scanning using FT, and resolution ratio is
30000, scanning rangem/z100~1000, width 2 is isolated；Second level and three-level mass spectrum are using data dependency scanning (data
Dependent scan, DDS), it chooses highest 3 peaks of upper level abundance and carries out collision induced dissociation (collision
Induced dissociation, CID) fragment scanning, normalization collision energy is 35%, detects fragment with ion trap dynode
Ion.
Above-mentioned UHPLC-MS detection method further includes the steps that wherein reference substance solution can contain by reference substance solution sample introduction
There are glycyrrhizin, isoliquiritigenin, enoxolone, liquiritin, isoliquiritin, liquorice chalcone B, celery sugar liquiritin, liquorice chalcone
A, glabridin, ononin, secondary liquorice chalcone, at least one of onocerin and camphor, preferably two or more, it is more excellent
5 kinds or more are selected, further preferably 8 kinds or more.Using above-mentioned reference substance solution sample introduction, be conducive to determine each group in Brown Mixtura sample
The identification divided.The preparation method of the reference substance solution can be with are as follows: each reference substance is accurately weighed, and every kind of control is made in solubilizer
Product concentration is 10-500 μ gmL-1Reference substance solution.Solvent can be selected from 50-100% methanol aqueous solution, 50-100% ethyl alcohol
Aqueous solution, 50-100% acetonitrile solution, preferably 80-100% methanol aqueous solution, 80-100% ethanol water, 80-100% second
Nitrile aqueous solution, more preferably methanol or acetonitrile.The concentration of every kind of reference substance solution can be 50-200 μ gmL-1, more preferably
For 80-150 μ gmL-1, more preferably 100 μ gmL-1.Using above-mentioned reference substance solution sample introduction, be conducive to Brown Mixtura
The identification of each component in sample.
Case is embodied, the present invention will be further described below:
One, instrument and material
1. key instrument
3000 Ultra Performance Liquid Chromatography instrument of DIONEX Ultimate and LTQ-Orbitrap XL mass spectrum are furnished with electron spray ion
Source (ESI), 2.1 work station of Xcalibur (Thermo Scientific company of the U.S.)；Sartorious BT 25S type ten
A ten thousandth electronic analytical balance (Beijing Sai Duolisi Instrument Ltd.)；KQ-100DE type numerical control ultrasonic cleaner (elder brother
Ultrasonic instrument Co., Ltd of mountain city)；Acquity UPLC ® BEH C18Column(2.1 mm × 100 mm, 1.7 μm,
Waters company, Milford, MA, USA), (U.S. Millipore is public for Millipore Synergy UV type ultrapure water machine
Department)；GracePureTM SPE C18- Low solid phase extraction column (500 mg/3 mL).
2. experimental material
Reference substance: glycyrrhizin, isoliquiritigenin, enoxolone, liquiritin, isoliquiritin, liquorice chalcone B, celery sugar liquiritin are sweet
Careless chalcone A, glabridin, ononin, secondary liquorice chalcone, onocerin and camphor.All reference substance purity are all larger than
98%。
Brown Mixtura is provided by Kun Yao Group Plc (specification: 100 pieces/bottle, lot number: Z53020718)；
Acetonitrile and methanol (mass spectrum grade, German Merck company), formic acid (chromatographic grade, German Merck company).
Sprague Dawley(SD) rat, male, weight (220 ~ 250g), be purchased from Beijing dimension tonneau China experimental animal skill
Art Co., Ltd, the capital credit number SCXK() 2012-0001.
Two, experimental method
1, the preparation of mixed reference substance solution
Take above-mentioned 13 reference substances appropriate respectively, accurately weighed, adding methanol that concentration is made is about 100 μ gmL-1Stock solution；
It is accurate respectively that draw each stock solution appropriate, add methanol constant volume in 5 mL volumetric flasks to get.
, test solution preparation
0.50 g of extracting liquorice piece is ground, and 10 mL70% methanol 20 min of ultrasound are added, and filtering takes subsequent filtrate, and 0.22 μm excessively micro-
Hole filter membrane is to get the first test solution.
, chromatographic condition
Chromatographic column: ACQUITY UHPLC BEH C18(1.7 μm, 2.1 × 100mm)；Mobile phase: 0.1% aqueous formic acid (A)-
Acetonitrile (B)；Gradient elution: 0 min, 5% B；10 min, 42% B；15 min, 46% B；20 min, 55% B；25 min, 65%
B；30 min, 100% B.Flow velocity is 0.3 mLmin-1；2 μ L of sample volume.
, Mass Spectrometry Conditions
Positive ion detection mode: capillary temperature: 350 °C；Sheath gas: 40.0 arb；Secondary air speed: 20.0 arb；Spray
Mist voltage: 4 kV；Capillary voltage: 25 V；Pipe lens voltage: 110 V.Sample carries out FS scanning using FT, and resolution ratio is
30000, scanning rangem/z100~1000, width 2 is isolated；Second level and three-level mass spectrum are using data dependency scanning (data
Dependent scan, DDS), it chooses highest 3 peaks of upper level abundance and carries out collision induced dissociation (collision
Induced dissociation, CID) fragment scanning, normalization collision energy is 35%, detects fragment with ion trap dynode
Ion.
, zoopery and sample acquire
SD rat 8 (each 4 of blank group, administration group), in 22 ~ 26 DEG C of room temperature, humidity is 40% ~ 70% for raising, and 12 h are round the clock more
In the animal housing replaced, adaptable fed one week before testing, tested preceding 12 h of fasting, whole process can't help water.
Dosage: 612.5 mgkg are pressed-1Gastric infusion, 2 times a day, each 2ml, continuous gavage 3 days.Blank group is given
Give the physiological saline of equivalent.
Plasma sample acquisition: respectively at 0.5,1,2 and 4 h times after the rat last time stomach-filling of blank group and administration group
Point eye socket takes 0.5 mL of blood, is placed in the anticoagulant EP pipe of heparin sodium, is uniformly mixed, and 3500rpm is centrifuged 10 min, merges 4 respectively
The supernatant at time point saves backup to get blank plasma and plasma containing drug in -20 DEG C of refrigerators.
, rat plasma sample processing method
Successively with 5 mL methanol and 5 mL water activated solid extraction columns, 1 mL blood plasma is then added, finally with 5 mL water and 3 mL
Methanol elution, collects meoh eluate, and at room temperature with being dried with nitrogen, residue is added 100 μ L, 10% acetonitrile solution and redissolves, vortex 4
It is centrifuged 20 min(14000 rpm after min, 4 DEG C), Aspirate supernatant is to get the second test solution.
Three, experimental result
Detect to obtain the accurate relative molecular mass of each chemical component, retention time in Brown Mixtura by analyzing UHPLC-MS
And Information in Mass Spectra, and combine and extract ion flow graph and carry out chemical component Structural Identification with reference substance, pertinent literature data.Most
Eventually, chemical component 55 are identified from Brown Mixtura altogether；While it being moved from being identified in the rat plasma after administration in 26 blood
Row ingredient, including 20 original shape ingredients and 6 metabolites, the result is shown in Figure 1 and table 1.
1 UHPLC-LTQ-Orbitrap-MS of table produces chemical component in Brown Mixtura and plasma sample prototype and metabolism
The identification and analysis of object
F and MFRespectively flavone compound and its metabolite；T and MTRespectively terpenoid and its metabolite；A is
Alkaloid compound；△It is compared with reference substance.
Four, methodological study
This research has carried out the investigation of more system to the precision, repeatability and stability of this method.
1, precision
Brown Mixtura sample is taken, the first test solution is prepared according to the regulation of the first sample solution preparation method, according to
Analysis method continuous sample introduction 6 times as defined in chromatographic condition are identified in calculating Brown Mixtura using liquiritin chromatographic peak as reference
The relative retention time and relative peak area of 55 chemical component chromatographic peaks, and its RSD is evaluated.6 sample introductions, 55 colors
The relative retention time RSD of spectral peak is lower than 0.5%, and relative peak area RSD is lower than 2.0%.
The plasma sample for taking Brown Mixtura Metabolite, according to the regulation of the second sample solution preparation method preparation the
Two test solutions, using liquiritin chromatographic peak as reference, are calculated according to analysis method continuous sample introduction 6 times as defined in chromatographic condition
The relative retention time of 26 chemical component chromatographic peaks of the plasma sample identification of Brown Mixtura Metabolite and opposite peak face
Product, and its RSD is evaluated.The relative retention time RSD of 6 sample introductions, 26 chromatographic peaks is lower than 0.5%, relative peak area RSD
Lower than 2.0%.
The above result shows that method precision is good.
2, repeated
Brown Mixtura sample is taken, it is molten to prepare 6 part of first test sample in parallel according to the regulation of the first sample solution preparation method
Liquid distinguishes sample introduction according to analysis method as defined in chromatographic condition, using liquiritin chromatographic peak as reference, calculates and reflects in Brown Mixtura
The relative retention time and relative peak area of 55 fixed chemical component chromatographic peaks, and its RSD is evaluated.6 sample introductions 55
The relative retention time RSD of a chromatographic peak is lower than 0.5%, and relative peak area RSD is lower than 2.0%.
The plasma sample for taking Brown Mixtura Metabolite is made in parallel according to the regulation of the second sample solution preparation method
Standby 6 part of second test solution distinguishes sample introduction according to analysis method as defined in chromatographic condition, using liquiritin chromatographic peak as reference,
Calculate the relative retention time of 26 chemical component chromatographic peaks of the plasma sample identification of Brown Mixtura Metabolite and opposite
Peak area, and its RSD is evaluated.The relative retention time RSD of 6 sample introductions, 26 chromatographic peaks is lower than 0.5%, opposite peak face
Product RSD is lower than 2.0%.
The above result shows that method repeatability is good.
3, stability
Brown Mixtura sample is taken, the first test solution is prepared according to the regulation of the first sample solution preparation method, respectively
0,3,6,9,12,24 hour after preparation according to analysis method sample introduction as defined in chromatographic condition, be ginseng with liquiritin chromatographic peak
According to, the relative retention time and relative peak area of the 55 chemical component chromatographic peaks identified in calculating Brown Mixtura, and to it
RSD is evaluated.The relative retention time RSD of 6 sample introductions, 55 chromatographic peaks is lower than 0.5%, and relative peak area RSD is lower than 2.0%.
The plasma sample for taking Brown Mixtura Metabolite, according to the regulation of the second sample solution preparation method preparation the
Two test solutions, 0,3,6,9,12,24 hour after preparation according to analysis method sample introduction as defined in chromatographic condition,
Using liquiritin chromatographic peak as reference, 26 chemical component chromatographic peaks of the plasma sample identification of Brown Mixtura Metabolite are calculated
Relative retention time and relative peak area, and its RSD is evaluated.The relative retention time of 6 sample introductions, 26 chromatographic peaks
RSD is lower than 0.5%, and relative peak area RSD is lower than 2.0%.
The above result shows that method is good in 24 hours internal stabilities.
Claims (10)
1. a kind of method of chemical component in UHPLC method detection Brown Mixtura, it is characterised in that the UHPLC method detection is multiple
The method of chemical component includes that detection Brown Mixtura chemical component and detection Brown Mixtura blood plasma are metabolized in square licorice tablet
Point, specifically includes the following steps:
A, prepared by test solution:
Brown Mixtura is taken to carry out that the first test solution is prepared；The blood plasma of Brown Mixtura Metabolite is taken to be prepared
Obtain the second test solution；
Preparation method specifically:
1) the first test solution: taking Brown Mixtura, crushes, and 10 ~ 40 times of glycyrrhiza compound sheet weight of organic solvent is added and mentions
It takes, extracting solution filtering, filtrate is the first test solution；
2) the second test solution: by the solid-phase extraction column after being activated on the blood plasma containing Brown Mixtura Metabolite, successively
It being eluted with water and methanol, collects meoh eluate, volatilize, the acetonitrile solution that percentage by volume 10% is added redissolves, vortex 3 ~
10 ~ 30min is centrifuged after 5min, taking supernatant is the second test solution；
B, prepared by reference substance solution: reference substance solution is glycyrrhizin, isoliquiritigenin, enoxolone, liquiritin, isoliquiritin, sweet
Careless chalcone B, celery sugar liquiritin, licochalcone A, glabridin, ononin, secondary liquorice chalcone, onocerin and camphor tree
One or more of brain reference substance；
Preparation method be each reference substance is accurately weighed, solubilizer be made every kind of reference substance concentration be 10 ~ 500 μ g mL-1 pair
According to product solution；The solvent is 50 ~ 100% methanol aqueous solution of percentage by volume, 50 ~ 100% ethanol waters or 50 ~ 100% second
Nitrile aqueous solution；
C, UHPLC is measured:
UHPLC chromatographic condition is as follows:
Chromatographic column: C18Column；Mobile phase: 0.1% aqueous formic acid (A)-acetonitrile (B)；Gradient elution: 0 min, 5% B；10 min,
42% B；15 min, 46% B；20 min, 55% B；25 min, 65% B；30 min, 100% B, flow velocity are 0.3 mLmin-1；Sample volume 0.5-20 μ L.
2. the method for chemical component in UHPLC method detection Brown Mixtura according to claim 1, it is characterised in that A step
It is rapid 1) described in organic solvent be 50 ~ 100% methanol aqueous solution of percentage by volume, 50 ~ 100% ethanol waters or 50 ~ 100%
Acetonitrile solution.
3. the method for chemical component in UHPLC method detection Brown Mixtura according to claim 1, it is characterised in that A step
It is rapid 1) described in organic solvent be 60 ~ 80% methanol aqueous solution of percentage by volume, 60 ~ 80% ethanol waters or 60 ~ 80% acetonitriles
Aqueous solution.
4. the method for chemical component in UHPLC method detection Brown Mixtura according to claim 1, it is characterised in that A step
It is rapid 1) described in organic solvent be 70% methanol solution of percentage by volume.
5. the method for chemical component in UHPLC method detection Brown Mixtura according to claim 1, it is characterised in that A step
It is rapid 2) described in the blood plasma containing Brown Mixtura Metabolite be to be prepared by the following, specially animal or population clothes
Brown Mixtura is given, blood sampling is placed in the anticoagulant EP pipe of heparin sodium after drug enters blood, is uniformly mixed, and centrifugation takes supernatant to obtain
It arrives.
6. the method for chemical component in UHPLC method detection Brown Mixtura according to claim 1, it is characterised in that A step
It is rapid 2) described in activation solid-phase extraction column be successively with first alcohol and water activate.
7. the method for chemical component in UHPLC method detection Brown Mixtura according to claim 1, it is characterised in that A step
It is rapid 2) described in centrifugal condition be 4 DEG C of temperature, revolving speed 14000rpm.
8. the method for chemical component in UHPLC method detection Brown Mixtura according to claim 1, it is characterised in that B step
Organic solvent described in rapid is 80 ~ 100% methanol aqueous solution of percentage by volume, 80 ~ 100% ethanol waters or 80 ~ 100% acetonitriles
Aqueous solution.
9. the method for chemical component in UHPLC method detection Brown Mixtura according to claim 1, it is characterised in that also wrap
Mass spectroscopy detection step is included, i.e., the method for chemical component is the detection of UHPLC-MS method in described UHPLC method detection Brown Mixtura.
10. the method for chemical component in UHPLC method detection Brown Mixtura according to claim 1, it is characterised in that matter
Spectral condition is as follows: positive ion detection mode: capillary temperature: 350 °C；Sheath gas: 40.0 arb；Secondary air speed: 20.0
arb；Spray voltage: 4 kV；Capillary voltage: 25 V；Pipe lens voltage: 110 V；Sample carries out FS scanning using FT, differentiates
Rate is 30000, scanning rangem/z100~1000, width 2 is isolated；Second level and three-level mass spectrum are scanned using data dependency
(data dependent scan, DDS) chooses highest 3 peaks of upper level abundance and carries out collision induced dissociation (collision
Induced dissociation, CID) fragment scanning, normalization collision energy is 35%, detects fragment with ion trap dynode
Ion.
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| CN109342609B (en) * | 2018-12-21 | 2021-04-20 | 国药控股星鲨制药（厦门）有限公司 | Method for identifying chemical components of traditional Chinese medicine fluid extract of compound three-dimensional calcium D-pantothenate syrup |
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